Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for &quot;prion-like&quot; conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response.

pbio.1002579.g002: RT-QuIC shows widespread detection of misfolded prion seeds beyond levels detected using IHC.(a) ThT fluorescence readout over time during the RT-QuIC assay. Each solid line represents a GSS/101LL brain region, whereas each dotted line represents a NBH/101LL brain region. These data are comprised of the averages from triplicate RT-QuIC reactions from three separate repeat experiments collected from four separate GSS/101LL dissected brains and five NBH/101LL dissected brains all at the terminal stage of disease (291.1 ± 5.3 dpi). The different brain regions are colour coded to illustrate the brain stem (red), thalamus (blue), cerebellum (purple), and cortex (green) for both the GSS/101LL and NBH/101LL samples. (b) RT-QuIC ThT fluorescence readout at 48 h (respective to cycle 190 in the RT-QuIC assay) using samples with or without PK exposure. ThT fluorescence increases are observed in each PK-exposed brain region of GSS/101LL mice (n = 4), showing that the misfolded PrP responsible for the seeding event has obtained a PK-resistant conformation in all GSS/101LL brain regions. No increase in ThT fluorescence was observed in NBH/101LL control brain regions (n = 5), demonstrating the specificity of prion seeding ability in GSS/101LL brain regions. GSS/101LL samples are presented as red (brain stem), blue (thalamus), purple (cerebellum), or green (cortex), and region-matched NBH/101LL controls are plotted in the same columns as grey open dots. These data are comprised of triplicate RT-QuIC reactions for each brain region of each animal tested.

Mentions:
We hypothesised that misfolded prion seeds would also be restricted to the specific brain regions associated with IHC-detectable misfolded PrP and that these brain regions would specifically undergo neurodegeneration, whereas those with no immunopositive PrP deposits would contain no prion seeds and remain free of neurodegeneration. In order to test this hypothesis, four brain regions were assessed for the presence of prion seeds as defined by their ability to act as seeds in the RT-QuIC assay and generate a ThT-positive signal. These were two IHC-positive regions the brain stem (between Bregma -6 to -8) and thalamus (between Bregma -1 to -3) and two IHC-negative regions the cerebellum (between Bregma -6 to -8) and cerebral cortex (between Bregma -1 to -3) (henceforth referred to as cerebellum, brain stem, thalamus, and cortex). All of these brain regions from GSS/101LL mice, when used in the RT-QuIC assay, elicited an increased ThT fluorescence not observed in uninfected NBH controls (Fig 2A). Thus, the increased level of fibril formation was specific to prion infection and demonstrated the presence of prion seeds in each brain region tested. To assess whether the prion seeds detected in these brain regions represented a protease-resistant conformational rearrangement of PrP [21], proteinase K (PK) digestion was performed on all samples of brain regions prior to their inclusion in the RT-QuIC assay. In all regions from prion-infected brains exposed to PK, prion seeds remained detectable, but PK-resistant prion seeds were not observed in any region of age-matched uninfected NBH controls (Fig 2B). To confirm the widespread appearance of prion seeds, we have tested these same brain regions that gave positive RT-QuIC detection in a different assay: the protein misfolded cyclic amplification (PMCA) assay [22]. We show that, similar to RT-QuIC, all brain regions from GSS/101LL tested were capable of seeding the PMCA reaction (S1 Fig), conclusively demonstrating the widespread accumulation of misfolded PrP, which can act as prion seeds in all brain regions tested in GSS/101LL mice.

pbio.1002579.g002: RT-QuIC shows widespread detection of misfolded prion seeds beyond levels detected using IHC.(a) ThT fluorescence readout over time during the RT-QuIC assay. Each solid line represents a GSS/101LL brain region, whereas each dotted line represents a NBH/101LL brain region. These data are comprised of the averages from triplicate RT-QuIC reactions from three separate repeat experiments collected from four separate GSS/101LL dissected brains and five NBH/101LL dissected brains all at the terminal stage of disease (291.1 ± 5.3 dpi). The different brain regions are colour coded to illustrate the brain stem (red), thalamus (blue), cerebellum (purple), and cortex (green) for both the GSS/101LL and NBH/101LL samples. (b) RT-QuIC ThT fluorescence readout at 48 h (respective to cycle 190 in the RT-QuIC assay) using samples with or without PK exposure. ThT fluorescence increases are observed in each PK-exposed brain region of GSS/101LL mice (n = 4), showing that the misfolded PrP responsible for the seeding event has obtained a PK-resistant conformation in all GSS/101LL brain regions. No increase in ThT fluorescence was observed in NBH/101LL control brain regions (n = 5), demonstrating the specificity of prion seeding ability in GSS/101LL brain regions. GSS/101LL samples are presented as red (brain stem), blue (thalamus), purple (cerebellum), or green (cortex), and region-matched NBH/101LL controls are plotted in the same columns as grey open dots. These data are comprised of triplicate RT-QuIC reactions for each brain region of each animal tested.

Mentions:
We hypothesised that misfolded prion seeds would also be restricted to the specific brain regions associated with IHC-detectable misfolded PrP and that these brain regions would specifically undergo neurodegeneration, whereas those with no immunopositive PrP deposits would contain no prion seeds and remain free of neurodegeneration. In order to test this hypothesis, four brain regions were assessed for the presence of prion seeds as defined by their ability to act as seeds in the RT-QuIC assay and generate a ThT-positive signal. These were two IHC-positive regions the brain stem (between Bregma -6 to -8) and thalamus (between Bregma -1 to -3) and two IHC-negative regions the cerebellum (between Bregma -6 to -8) and cerebral cortex (between Bregma -1 to -3) (henceforth referred to as cerebellum, brain stem, thalamus, and cortex). All of these brain regions from GSS/101LL mice, when used in the RT-QuIC assay, elicited an increased ThT fluorescence not observed in uninfected NBH controls (Fig 2A). Thus, the increased level of fibril formation was specific to prion infection and demonstrated the presence of prion seeds in each brain region tested. To assess whether the prion seeds detected in these brain regions represented a protease-resistant conformational rearrangement of PrP [21], proteinase K (PK) digestion was performed on all samples of brain regions prior to their inclusion in the RT-QuIC assay. In all regions from prion-infected brains exposed to PK, prion seeds remained detectable, but PK-resistant prion seeds were not observed in any region of age-matched uninfected NBH controls (Fig 2B). To confirm the widespread appearance of prion seeds, we have tested these same brain regions that gave positive RT-QuIC detection in a different assay: the protein misfolded cyclic amplification (PMCA) assay [22]. We show that, similar to RT-QuIC, all brain regions from GSS/101LL tested were capable of seeding the PMCA reaction (S1 Fig), conclusively demonstrating the widespread accumulation of misfolded PrP, which can act as prion seeds in all brain regions tested in GSS/101LL mice.

Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for &quot;prion-like&quot; conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response.